Interleukin (IL)-22 regulates the pathogenesis of autoimmune diseases. The role of IL-22+ T-cells in the pathogenesis of rheumatoid arthritis (RA) is unclear. This study aimed at examining the levels of plasma IL-22 and the frequency of IL-22+ CD4+ T-cells in patients with RA.
A total of 30 RA patients and 18 gender- and age-matched healthy controls were recruited. Their peripheral blood mononuclear cells were isolated and stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 6 h. The frequency of IL-22+, interferon (IFN)-γ+ and IL-17A+ CD4+ T-cells was characterized by flow cytometry. The levels of plasma IFN-γ, IL-17A and IL-22, serum C-reactive protein (CRP), rheumatoid factor (RF), anticyclic citrullinated peptide antibody (CCP) and erythrocyte sedimentation rate (ESR) were measured.
The frequency of IFNγ–IL-17A–IL-22+, IFNγ–IL-17A+IL-22+, and IFNγ+IL-17A–IL-22+ T-cells in CD4+ T-cells and the levels of plasma IFNγ, IL-17 and IL-22 in RA patients were significant higher than those in healthy controls. The percentages of IL-17A+IL-22+CD4+ T-cells were correlated positively with the frequency of Th22 or Th17 cells in the RA patients. The percentages of IL-22+CD4+ T-cells were correlated positively with the values of disease activity score (DAS28) in the RA patients. The percentages of Th22 cells were correlated positively with the levels of plasma IL-22 in the RA patients.
Our data suggest that IL-22+CD4+ T-cells may contribute to the pathogenesis of RA and that therapeutic targeting of IL-22 may be valuable for the intervention of RA.
Rheumatoid arthritis (RA) is a chronic autoimmune disease and is characterized by severe joint deformities due to bony erosions and tendon damage. The chronic inflammatory process is regulated by a complex cytokine network and matrix metalloproteinases (MMPs). The roles of various cytokines in the development and progression of RA have been extensively studied, which have resulted in the development of new therapies for the intervention of RA. Therefore, continual studies of new cytokines in the pathogenesis of RA will be of great significance.
Interleukin (IL)-22 is one member of the IL-10 family and has 22% homology to IL-10. IL-22 is produced by several types of cells, including activated CD4+ T-cells, natural killer (NK) cells, NKT cells and lymphoid tissue inducer cells.[3-5] IL-22 has many functions, such as protection against tissue damage and regulating inflammation and autoimmunity.[6-8] Previous studies have shown that IL-22 can induce the expression of anti-inflammatory proteins, such as follistatin and IL-11, and induce the expression of pro-inflammatory cytokines, such as IL-6, chemokines and acute-phase reactants.[9, 10] Indeed, elevated levels of plasma IL-22 are detected in patients with psoriasis and correlated with disease severity, while the levels of plasma IL-22 are reduced following anti-psoriatic therapy.[7, 11] In addition, higher levels of serum IL-22 are detected in patients with Crohn's disease and are associated with up-regulated tumor necrosis factor alpha (TNF-α) and IL-8 expression in intestinal epithelial cells. Similarly, increased concentrations of serum IL-22 are also observed and associated with disease severity in patients with RA.[13, 14] There are different subsets of activated CD4+ T-cells, such as interferon-gamma (IFNγ)-IL-17–IL-22+ (Th22), IFNγ+IL-17–IL-22+ (IL-22+ Th1), and IFNγ-IL-17+IL-22+ (IL-22+ Th17) that produce IL-22 and contribute to the pathogenesis of pro-inflammatory autoimmune diseases. However, which type of IL-22+ CD4+ T-cells is associated with elevated levels of serum IL-22 has not been clarified in patients with RA.
The functional differentiation of Th22 cells is regulated by the aryl hydrocarbon receptor (AHR), a key transcription factor, and Th22 cells secrete IL-22, which can activate signal transduction and transcription 3 (STAT3). The functional differentiation of Th22 cells is regulated positively by IL-6, TNF-α and IL-23. Previous studies have shown that a higher frequency of Th22 is detected in patients with RA.[14, 18] Notably, IL-6 and IL-23 are crucial regulators of Th17 differentiation, and Th17 cells are important players in the pathogenesis of RA.[19, 20] In addition, the role of IFNγ+ Th1 cells in the pathogenesis of RA remains controversial.[21-23] Therefore, the frequency of peripheral blood IL-22+ Th1 and Th17 cells in patients with RA and their roles in the pathogenesis of RA have not been clarified. Moreover, how the relationship works among Th17, Th22 and Th1 cells in the development and progression of RA, and whether the frequency of Th17, Th22 and Th1 is associated with the disease severity in Chinese patients with RA, have not been clarified.
In this study, we characterized the frequency of peripheral blood Th22, Th17, Th1, IL-22+ Th17 and IL-22+ Th1 cells in 30 patients with new onset RA and 18 gender- and age-matched healthy controls, by flow cytometry analysis. Furthermore, we examined the concentrations of plasma IL-22, IL-17 and IFNγ in this population. In addition, we investigated the potential correlation between the frequency of different functional CD4+ T-cells and clinical disease severity in these RA patients. Our data suggest that IL-22+CD4+ T-cells may contribute to the pathogenesis of RA in Chinese patients.
Materials and Methods
A total of 30 patients with new onset RA were recruited from the inpatient service of the First Hospital of Jilin University between March and June of 2011. An additional 18 gender- and age-matched healthy subjects were recruited from the outpatient service at the same hospital. Individual patients with RA were diagnosed, according to the revised criteria for the classification of RA by the American College of Rheumatology. The degrees of disease activity in these patients were assessed using the disease activity score in 28 joints (DAS28), and a score ≥ 2.6 was defined active disease. Patients were excluded if they had myositis, systemic sclerosis, or other autoimmune diseases and if he/she had received immunosuppressive therapy or glucocorticoid therapy within the past 6 months. Written informed consent was obtained from each participant. The experimental protocol was established according to the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of Jilin University.
The baseline demographic and clinical data were collected from hospital records and reviewed by experienced physicians. The data included age, sex and current medications. Routine laboratory investigation included full blood count, serum C-reactive protein (CRP) levels, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (CCP). The levels of serum CRP, ESR and RF were determined by scatter turbidimetry using a Siemens special protein analyzer (Siemens Healthcare Diagnostics Products, GmbH, Munich, Germany). The levels of serum CCP were determined by the chemoluminescence microparticle immune method using a specific reagent (Abbott, GmbH, Wiesbaden, Germany).
Isolation and stimulation of PBMCs
Fasting peripheral venous blood samples were collected from individual participants and peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). PBMCs at 106/mL were stimulated in duplicate with phorbol 12-myristate 13-acetate (PMA, 1 μg/mL) and ionomycin (50 μg/mL; Sigma, St. Louis, MO, USA) in 10% human sera (AB type) in Roswell Park Memorial Institute (RPMI) 1640 medium at 37°C in a humidified incubator of 95% air and 5% carbon dioxide for 4 h and cultured for another 2 h in the presence of brefeldin A (BFA, 0.5 μg/mL; Sigma). The same cells were cultured in medium alone and used as negative controls.
Flow cytometry analysis
The stimulated PBMCs were harvested and stained with PerCP-anti-CD4 (Becton Dickinson, San Diego, CA, USA), fixed with 4% paraformaldehyde at room temperature (RT) for 30 min and then permeabilized with 0.5% saponin in 10% fetal bovine serum (FBS) in PBS (30 min at RT). After being washed, the cells were stained with FITC-anti-IFN-γ, Alexa-Flour-anti-IL-17 (Becton Dickinson), and PE-anti-IL-22 (R&D Systems, Minneapolis, MN, USA). The frequency of cytokine+ T-cells was determined by flow cytometry analysis on a BD FACSCalibur (Becton Dickinson) using FlowJo software version 7.5.5 for Microsoft ( TreeStar, San Carlos, CA).
The concentrations of plasma IFN-γ, IL-17 and IL-22 in individual participants were determined by enzyme-linked immunosorbent assay (ELISA) using specific cytokine kits, according to the manufacturer's instructions (R&D Systems).
All data are expressed as individual values, median and range of each group of subjects. The difference between groups was analyzed using the Mann–Whitney U-test. The potential correlation between variables was analyzed by the Spearman rank correlation test. All statistical tests were performed using SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA). A two-sided P-value of < 0.05 was considered statistically significant.
A high frequency of peripheral blood IL-22+CD4+ T-cells in the patients with RA
To examine the frequency of different subsets of CD4+ T-cells, a total of 30 patients with new-onset RA and 18 gender- and age-matched healthy subjects were recruited, and their demographic and clinical characteristics are shown in Table 1. As expected, there was no significant difference in distribution of gender and age, and the white blood cell (WBC) counts between the RA patients and healthy controls. In contrast, the levels of serum RF, CCP, ESR and CRP in the RA patients were significantly higher than that in the healthy controls.
Table 1. Clinical and laboratory characteristics of subjects
Healthy controls (n = 18)
RA patients (n = 30)
Data shown are median (range) or real case numbers. ND, undetectable; RF, rheumatoid factor; CCP, anticyclic citrullinated peptide antibody; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; DAS28, disease activity score of 28 joints; WBC, white blood cell count. The normal ranges of RF, CCP, CRP and ESR are 0–15 IU/mL, 0–25 U/mL, 0–15 mg/L and 0–5 mm/h, respectively. P < 0.05 versus the controls.
WBC (× 109/L)
Flow cytometry analysis indicated that the frequency of peripheral blood Th22 and Th17 cells in the patients with RA was significantly higher than those in the controls (Fig. 1). Further analysis revealed that the frequency of peripheral blood IFNγ+IL-17–IL-22+ and IFNγ–IL-17+IL-22+ CD4+ T-cells in the RA patients was significantly higher than those in the healthy controls (Fig. 2). Therefore, a higher frequency of peripheral blood IL-22+CD4+ T-cells existed in the RA patients.
Percentages of IFNγ–IL-17+IL-22+ CD4+ T-cells are correlated with frequency of Th22 or Th17 cells in RA patients
To investigate the potential relationship among IL-22, IFN-γ or IL-17 producing CD4+ T-cells, we performed a correlation analysis. We found that the percentages of IFNγ–IL-17+IL-22+ CD4+ T-cells were correlated positively with the frequency of Th22 (r = 0.2307, P = 0.0062, Fig. 3) or Th17 (r = 0.1411, P = 0.0488; Fig. 3) cells in the RA patients. However, there was no significant association among Th22, Th17, Th1, IFNγ–IL-17+IL-22+ and IFNγ+IL-17–IL-22+ CD4+ T-cells in this population (data not shown).
Increased frequency of Th22 cells is correlated with elevated levels of serum IL-22 in RA patients
We further tested the concentrations of plasma IL-22, IL-17 and IFN-γ in the RA patients and healthy controls by ELISA. We found that the concentrations of plasma IL-22, IL-17 and IFN-γ were significantly higher in RA patients than that in controls (Fig. 4). The levels of plasma IL-22 in the RA patients were correlated positively with the percentages of their peripheral blood Th22 cells (r = 0.8269, P < 0.0001, Fig. 4). However, there was no significant correlation between the levels of plasma IL-22 and the frequency of IFNγ+IL-17–IL-22+CD4+ T-cells or the percentages of IFNγ–IL-17+IL-22+CD4+ T-cells in these patients. Similarly, there was no significant correlation of the concentrations of plasma IFNγ with the frequency of Th1 cells, and no significant correlation between the levels of plasma IL-17 and the frequency of Th17 cells in these patients.
Percentages of peripheral blood IL-22+CD4+ T-cells are correlated significantly with the degrees of disease severity in RA patients
To determine the potential role of IL-22+CD4+ T-cells in the development of RA, we analyzed the values of DAS28 in 16 RA patients. We found that DAS28 values were correlated positively with the percentages of peripheral blood Th22, IFNγ–IL-17+IL-22+ and IFNγ+IL-17–IL-22+ CD4+ T-cells (Fig. 5). Furthermore, the percentages of peripheral blood Th22 or Th17 cells were correlated significantly with the values of ESR in 17 RA patients. In addition, the percentages of Th17 cells were correlated positively with the concentrations of serum RF in these RA patients. However, there was no statistically significant association between ESR values and the frequency of IFNγ–IL-17+IL-22+ and IFNγ+IL-17–IL-22+CD4+ T-cells in these RA patients. Similarly, there was no significant association between the frequency of IL-22+ CD4+ T cells and the levels of serum CCP and CRP in these patients.
IL-22 has been implicated in the pathogenesis of autoimmune inflammatory diseases. Previous evidence has shown that the frequency of peripheral blood IL-22+ CD4+ T-cells in patients with ankylosing spondylitis (AS) is significantly higher than those in healthy controls and that higher levels of serum IL-22 are associated with the development and progression of Crohn's disease and psoriasis.[7, 11, 12, 23] In this study, we examined the concentrations of plasma IL-22 in RA patients and healthy controls. We found that the levels of plasma IL-22 in RA patients were significantly higher than those in the healthy controls. These results suggest that IL-22 may contribute to the pathogenesis of RA in Chinese patients. Our data were consistent with a previous report that elevated levels of IL-22 were detected in the synovial tissues from patients with RA and that higher levels of IL-22R1 expression were observed in both the lining and sublining layers of rheumatoid synovium. It is possible that IL-22, through the STAT3, ERK1/2, and p38 MAKP pathways, stimulates synovial fibroblast proliferation and MCP-1 production, leading to inflammation.[13, 26]
Naïve CD4+ T-cells, after activation, can differentiate into Th1, Th2, Th17, Th22, Tfh and Treg, which produce specific cytokines. However, some activated CD4+ T-cells are IFNγ+IL-17–IL-22+, IFNγ–IL-17+IL-22+ or IFNγ–IL-17–IL-22+. In this study, we found that the frequency of peripheral blood Th17, IFNγ–IL-17–IL-22+ (Th22), IFNγ+IL-17–IL-22+ or IFNγ–IL-17+IL-22+, but not Th1 cells, in RA patients, was significantly higher than that in healthy controls. Our data were consistent with a previous report that shows a higher frequency of IL-22+CD4+ T-cells in RA patients and revealed that not only is there a higher frequency of Th22 cells, but also IL-22+ Th1 and Th17 cells in RA patients. Our findings suggest that all types of IL-22+CD4+ T-cells may play a role in the development of RA in Chinese patients. However, we did not detect a significant difference in the frequency of IL-22+CD4– cells between the RA patients and healthy controls. Interestingly, we found that the concentrations of plasma IL-22 were correlated positively with the frequency of Th22 cells, but not with IFNγ+IL-17–IL-22+ and IFNγ–IL-17+IL-22+ in RA patients. These, together with the relatively higher frequency of Th22 and the lack of significantly higher frequency of IL-22+CD4– cells in RA patients, suggest that Th22 cells may be the major source of plasma IL-22 in these patients. We are interested in further investigating whether IL-22 comes mainly from Th22 cells in RA patients. More importantly, we found that the frequency of Th22 and IL-17+IL-22+ CD4+ T-cells was correlated with DAS28 values and that the frequency of Th22 was also correlated positively with ESR values in RA patients. These data suggest that Th22 and IL-17+IL-22+ CD4+ T-cells may be crucial for the activity and progression of RA and that their frequency in peripheral blood may be a valuable biomarker of RA activity in Chinese patients.
Previous studies have shown that Th1 and IFNγ contribute to the pathogenesis of RA.[21-23] In this study, we detected significantly higher levels of plasma IFNγ in RA patients. However, we did not detect a significantly higher frequency of IFNγ+ Th1 cells in RA patients. The difference in the frequency of Th1 cells between our study and others may stem from different populations of patients with various genetic backgrounds. Notably, high levels of IFNγ can be produced by NK, activated CD8+ T-cells, and macrophages, which may explain the higher levels of IFNγ in RA patients.
Th17 and IL-17 have been associated with the pathogenesis of RA in humans. We not only detected a higher frequency of Th17, but also higher levels of plasma IL-17 in RA patients, consistent with previous reports.[19, 20] More importantly, we found that the frequency of Th17 cells was correlated positively with ESR values and the concentrations of RF in RA patients. Given that ESR and RF are valuable biomarkers of inflammation and autoimmunity, the positive correlation between the frequency of Th17 and ESR and RF values possibly indicates the important role of Th17 and IL-17 in the pathogenesis of RA. Interestingly, we found that the frequency of IL-17+IL-22+ CD4+ T-cells was correlated positively with the percentages of Th17 and Th22 in these patients. Furthermore, the frequency of IL-17+IL-22+ CD4+ T-cells was also correlated significantly with DAS28 values in RA patients. Given that IL-23-related signaling is crucial for the differentiation of Th17 and IL-22 expression in T-cells,[27, 28] it is possible that IL-17+IL-22+ CD4+ T-cells may differentiate to either Th17 or Th22 during the development of RA. Conceivably, therapeutic targeting of IL-23 and related signaling mat be a promising strategy for RA intervention. Finally, we recognized the variable frequencies of IL17+IL22+ CD4+ and IFN-γ+IL-22+ CD4+ T-cells in these patients. However, we did not detect any significant association between these two populations of CD4+ T-cells in these patients, even after eliminating those with lower frequency of each type of cells (data not shown). Given that IFN-γ has been shown to inhibit the development of Th17 cells, the lack of inverse correlation between these types of cells suggests that these patients may not develop a special type of T-cell immunity during the process of RA. The diverse T-cell immunity may be associated with varying genetic backgrounds, possible IL-23 receptor mutation status, or other unknown factors. We are interested in further investigating the dynamics of T-cell immunity during the process of RA and whether an individual's genetic background, such as MHC type and the status of IL-23R, can shape T-cell immunity in RA patients.
We recognized that our study had limitations of small sample size and the lack of detecting antigen-specific T-cell responses, as well as inflammation in the target tissues. Furthermore, our study only centered on one time point. Therefore, further longitudinal studies of antigen-specific T-cell immunity and inflammation in the target tissues in a relative bigger population are warranted.
In conclusion, our data indicated a higher frequency of IL-22+ CD4+ and Th17 cells in RA patients and that the frequency of Th22 cells was correlated with the degree of RA severity in this population. These data suggest that IL-22+ CD4+ T cells and Th17 cells contribute to the pathogenesis of RA and that the frequency of Th22 cells may be a valuable biomarker for the evaluation of disease severity in Chinese RA patients. Therefore, our findings may provide new insights into the pathogenesis of RA and aid in the design of new immunotherapies for RA patients.
We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. This study was supported by grants from the National Natural Science Foundation of China (No. 30972610 and 81273240), Jilin Province Science and Technology Agency (No. 200705128 and 20110716), the Health Department Research Projects in Jilin Province (2009Z054), Bethune B plan of Jilin University.